Method of diagnosing nephrotic syndrome

ABSTRACT

This invention provides organic biomolecule markers (e.g., proteins) useful for differentiating minimal change nephrotic syndrome (MCNS) from focal segmental glomerulosclerosis (FCS), membranous nephrothropy (MN), and membranoproliferative glomerulonephritis (MPGN). This invention also provides organic biomolecule markers useful for evaluating the therapeutic value of agents for treating kidney disease.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a non-provisional of, and claims the benefit of thepriority date of, U.S. application Ser. No. 60/315,774, filed Aug. 30,2001, the disclosure of which is incorporated herein by reference forall purposes.

BACKGROUND OF THE INVENTION

Idiopathic nephrotic syndromes due to primary glomerular diseasesinclude the minimal change nephrotic syndrome (MCNS), the focalsegmental glomerulosclerosis (FGS), the membranous nephropathy (MN), andthe membranoproliferative glomerulonephritis (MPGN). Patients with thesediseases fall into hypoproteinemia due to the loss of substantialamounts of protein in the urine during the nephrotic stage, and in orderto compensate for the loss, protein synthesis is accelerated in theliver.

The four disease types above are finally diagnosed by taking a sample ofthe kidney (renal biopsy), and testing the sample with a lightmicroscope and an electron microscope, and also testing the samplethrough the immunofluorescent antibody method. This diagnostic processis a large burden on the patient and requires time and labor for thehistological examination.

Therefore, there has been a demand for a diagnosis method for thenephrotic syndromes by using blood serum or urine, which are relativelyeasily available. The present invention aims at providing a newdiagnosis method. Through this method, the patient can be relieved ofthe biopsy. At the same time, this method enables the distinctionbetween the MCNS and the FGS, in which the clinical feature is similarto MCNS.

BRIEF SUMMARY OF THE INVENTION

The present invention provides, for the first time, sensitive and quickmethods and kits that can be used as an aid for diagnosis of MCNS bymeasuring markers that are differentially present in samples of a MCNSpatient and a subject who does not have MCNS (e.g., MN, FGS, IgAnephropathy, or MPGN patients). By monitoring the amount of one or moreof these markers, the methods and kits of the invention can determinethe subject's pathological status using minute quantities of crudesamples. In particular, it has been found that alpha-2-HS glycoproteinbeta chain, a protein detected from serum samples as a 5910 Da peak bySELDI mass spectrometry, is a marker for MCNS.

In one aspect, the invention provides methods for aiding the diagnosisof a kidney disease which comprises detecting alpha-2-HS glycoproteinbeta chain in a sample; and correlating the detection of the marker ormarkers with a probable diagnosis of a kidney disease. In oneembodiment, the detection is performed by any one of a variety ofimmunoassays. In a second embodiment, the detection is performed bySELDI MS mass spectrometry.

In another aspect, the invention provides a kit for aiding in thediagnosis of kidney disease, wherein the kit comprises an MS SELDI probecomprising a surface-bound agent capable of binding an antibody; anantibody that specifically binds alpha-2-HS glycoprotein beta chain; andinstructions to detect alpha-2-HS glycoprotein beta chain by contactinga sample with the probe having the antibody bound thereto and detectingalpha-2-HS glycoprotein beta chain by mass spectrometry. In oneembodiment, the kit comprises a wash solution that removes unboundmaterial from the probe surface. In another embodiment, the kitcomprises instructions to correlate the detection of alpha-2-HSglycoprotein beta chain with nephrotic disease.

In still another aspect, the invention provides a kit for aiding in thediagnosis of kidney disease, wherein the kit comprises an ELISAsubstrate, an antibody that specifically binds alpha-2-HS glycoproteinbeta chain; and instructions to detect alpha-2-HS glycoprotein betachain by contacting a sample with the ELISA substrate having theantibody bound thereto and detecting alpha-2-HS glycoprotein beta chainby ELISA. In one embodiment, the kit comprises a second, labeledantibody that specifically binds alpha-2-HS glycoprotein beta chain. Inanother embodiment, the kit further comprises instructions to correlatethe detection of alpha-2-HS glycoprotein beta chain with nephroticdisease.

In one aspect, the invention provides methods for aiding a MCNSdiagnosis, which comprises determining a test amount of a marker in asample from a subject and determining whether the test amount is adiagnostic amount consistent with a diagnosis of MCNS. A test amount ofa single marker or a plurality of markers can be determined in thisaspect of the invention.

The markers can have any suitable characteristics, including anyapparent molecular weight. For example, these diagnostic markers includepolypeptides having an apparent molecular weight of about 2955.3 Da(DA-1), 6116.6 Da (DA-2), 5910.0 Da (DA-3), 2962.5 Da (DB-1), 6130.8 Da(DB-2), or 3161.5 Da (DB-3).

In yet another embodiment, a sample being tested is taken from asubject's blood, serum, urine, semen, seminal fluid, seminal plasma, ortissue extracts. Preferably, the sample is serum or urine.

In yet another embodiment, the methods for diagnosis comprisesdetermining a test amount of a marker in a sample using immunoassay orgas phase ion spectrometry wherein the markers are selected from thegroup consisting of polypeptides having an apparent molecular weight ofabout 2955.3 Da (DA-1), 6116.6 Da (DA-2), 5910.0 Da (DA-3), 2962.5 Da(DB-1), 6130.8 Da (DB-2), or 3161.5 Da (DB-3). Preferably, laserdesorption mass spectrometry is used.

In another aspect, the invention provides a method for detecting amarker, the method comprising contacting a sample from a subject with asubstrate comprising an adsorbent thereon under conditions to allowbinding between a marker and the adsorbent, wherein the marker is apolypeptide which is differentially present in samples of a MCNS and asubject who does not have MCNS (e.g., MN, FGS, IgA nephropathy, or MPGNpatients), and detecting the marker bound to the adsorbent by gas phaseion spectrometry.

In yet another embodiment, the method further comprises determining thetest amount of the marker bound on the probe substrate, and determiningwhether the test amount is a diagnostic amount consistent with adiagnosis of MCNS.

In yet another aspect, the invention provides a method for detecting amarker in a sample, the method comprising: providing an antibody thatspecifically binds to the marker, wherein the marker is a polypeptidewhich is differentially present in samples of a MCNS patient and asubject who does not have MCNS (e.g., a MN, FGS, IgA nephropathy, orMPGN patient), and contacting the sample with the antibody, anddetecting the presence of a complex of the antibody bound to the marker.Markers that are differentially present in samples from MCNS patientsinclude polypeptides having an apparent molecular weight of about 2955.3Da (DA-1), 6116.6 Da (DA-2), 5910.0 Da (DA-3), 2962.5 Da (DB-1), 6130.8Da (DB-2), or 3161.5 Da (DB-3).

In yet another embodiment, the method further comprises determining thetest amount of the marker bound on the probe substrate, and determiningwhether the test amount is a diagnostic amount consistent with adiagnosis of MCNS.

In yet another aspect, the invention provides a kit for aiding adiagnosis of MCNS, wherein the kit comprises a substrate comprising anadsorbent thereon, wherein the adsorbent is suitable for binding amarker and a washing solution or instructions for making a washingsolution, wherein the combination of the adsorbent and the washingsolution allows detection of the marker using gas phase ionspectrometry. The kit is capable of allowing determination of a testamount of a marker, wherein the marker is a polypeptide which isdifferentially present in samples of a MCNS patient and a subject whodoes not have MCNS (e.g., a MN, FGS, IgA nephropathy, or MPGN patient).

In one embodiment, the substrate in the kit is in the form of a probewhich is removably insertable into a gas phase ion spectrometer. Inanother embodiment, the kit further comprises another substrate whichcan be used together with the substrate comprising the adsorbent to forma probe which is removably insertable into a gas phase ion spectrometer.

In another embodiment, the kit further comprises instructions forsuitable operational parameters.

In yet another embodiment, the substrate comprises a hydrophobic groupand an anionic group as an adsorbent. In yet another embodiment, thesubstrate comprises a hydrophobic group as an adsorbent. In yet anotherembodiment, the substrate comprises a metal chelating group. In yetanother embodiment, the substrate comprises a metal chelating groupcomplexed with a metal ion as an adsorbent. In yet another embodiment,the substrate comprises an antibody that specifically binds to a markeras an adsorbent. In yet another embodiment, the washing solution is anaqueous solution.

In yet another embodiment, the kit comprises an antibody thatspecifically binds to the marker, and a detection reagent. Optionally,the antibody can be immobilized on a solid support.

In yet another embodiment, the kits can further comprise a standardindicating a diagnostic amount of the marker.

While the absolute identity of many markers is not yet known, suchknowledge is not necessary to measure them in a patient sample, becausethey are sufficiently characterized by, e.g., mass and by affinitycharacteristics. It is noted that molecular weight and bindingproperties are characteristic properties of these markers and notlimitations on means of detection or isolation. Furthermore, using themethods described herein or other methods known in the art, the absoluteidentity of the markers can be determined.

The present invention provides a method for evaluating the progress ofkidney disease and the therapeutic value of agents used to treat kidneydisease by comparing the results of measurement of markers conductedwith samples taken from the same patient before and after MCNStreatment. In the measurement graph, there is a high peak and low peakafter treatment. By using these peaks as markers, it is possible toevaluate the progress of the disease and the therapeutic value of agentsused to treat the disease.

The markers can have any suitable characteristics, including anyapparent molecular weight. For example, these therapeutic markersinclude polypeptides having an apparent molecular weight of about 2952.3Da (TA-1), 5910.0 Da (TA-2), 5922.6 Da (TB-1), 10224.4 Da (TB-2),10793.3 Da (TB-3), 13672.1 Da (TC-1), 13980.1 Da (TC-2), 13895.6 Da(TC-3), 13788.5 Da (TC-4), or 13965.4 Da (TC-5).

In another embodiment, the sample being tested is taken from a subject'sblood, serum, urine, semen, seminal fluid, seminal plasma, or tissueextracts. Preferably, the sample is serum or urine.

In yet another embodiment, the methods for evaluating the progress ofkidney disease and the therapeutic value of agents used to treat kidneydisease comprises determining a test amount of a marker in a sampleusing immunoassay or gas phase ion spectrometry wherein the markers areselected from the group consisting of polypeptides having an apparentmolecular weight of about 2952.3 Da (TA-1), 5910.0 Da (TA-2), 5922.6 Da(TB-1), 10224.4 Da (TB-2), 10793.3 Da (TB-3), 13672.1 Da (TC-1), 13980.1Da (TC-2), 13895.6 Da (TC-3), 13788.5 Da (TC-4), or 13965.4 Da (TC-5).Preferably, laser desorption mass spectrometry is used.

In yet another aspect, the invention provides a kit for aiding inevaluating the progress of the disease and the therapeutic value ofagents used to treat kidney disease, wherein the kit comprises asubstrate comprising an adsorbent thereon, wherein the adsorbent issuitable for binding a marker and a washing solution or instructions formaking a washing solution, wherein the combination of the adsorbent andthe washing solution allows detection of the marker using gas phase ionspectrometry. The kit is capable of allowing determination of a testamount of a marker, wherein the marker is a polypeptide which isdifferentially present in samples of a kidney disease patient before andafter treatment.

In one embodiment, the substrate in the kit is in the form of a probewhich is removably insertable into a gas phase ion spectrometer. Inanother embodiment, the kit further comprises another substrate whichcan be used together with the substrate comprising the adsorbent to forma probe which is removably insertable into a gas phase ion spectrometer.

In another embodiment, the kit further comprises instructions forsuitable operational parameters.

In yet another embodiment, the substrate comprises a hydrophobic groupand an anionic group as an adsorbent. In yet another embodiment, thesubstrate comprises a hydrophobic group as an adsorbent. In yet anotherembodiment, the substrate comprises a metal chelating group. In yetanother embodiment, the substrate comprises a metal chelating groupcomplexed with a metal ion as an adsorbent. In yet another embodiment,the substrate comprises an antibody that specifically binds to a markeras an adsorbent. In yet another embodiment, the washing solution is anaqueous solution.

In yet another embodiment, the kit comprises an antibody thatspecifically binds to the marker, and a detection reagent. Optionally,the antibody can be immobilized on a solid support.

In yet another embodiment, the kits can further comprise a standardindicating a treatment amount of the marker.

While the absolute identity of many markers is not yet known, suchknowledge is not necessary to measure them in a patient sample, becausethey are sufficiently characterized by, e.g., mass and by affinitycharacteristics. It is noted that molecular weight and bindingproperties are characteristic properties of these markers and notlimitations on means of detection or isolation. Furthermore, using themethods described herein or other methods known in the art, the absoluteidentity of the markers can be determined.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph comparing four examples of MCNS with four examples ofmembranous nephropathy (MN), two examples of focal segmentalglomerulosclerosis (FGS), two examples of IgA nephropathy, and twoexamples of membranoproliferative glomerulonephritis (MPGN). In FIG. 1A,the MCNS graph has a peak at 2955.3 Da (DA-1).

FIG. 1B is an expanded view of a representative example in FIG. 1A.

FIG. 2A is a graph comparing four examples of MCNS with four examples ofMN, two examples of FGS, two examples of IgA nephropathy and twoexamples of MPGN. In FIG. 2A, the MCNS graph has a peak at 6116.6 Da(DA-2).

FIG. 2B is an expanded view of a representative example in FIG. 2A.

FIG. 3A is a graph comparing four examples of MCNS with four examples ofMN, two examples of FGS, two examples of IgA nephropathy and twoexamples of MPGN. In FIG. 3A, the MCNS graph has a peak at 5910 Da(DA-3).

FIG. 3B is an expanded view of a representative example in FIG. 3A.

FIG. 4A is a graph comparing four examples of MCNS with four examples ofMN, two examples of FGS, two examples of IgA nephropathy and twoexamples of MPGN. In FIG. 4A, the MCNS graph has peaks at 2962.5 Da(DB-1) and 3161.5 Da (DB-3).

FIG. 4B is an expanded view of a representative example relating to thepeak at 2962.5 Da (DB-1) in FIG. 4A.

FIG. 4C is an expanded view of a representative example relating to thepeak at 3161.5 Da (DB-3) in FIG. 4A.

FIG. 5A is a graph comparing four examples of MCNS with four examples ofMN, two examples of FGS, two examples of IgA nephropathy and twoexamples of MPGN. In FIG. 5A, the MCNS graph has a peak at 6130.8 Da(DB-2).

FIG. 5B is an expanded view of a representative example in FIG. 5A.

FIG. 6 is a graph comparing values before and after MCNS treatment.Samples of serum, urine and peripheral blood lymphocyte (PBNC) wereused. In FIG. 6, the peak at 2952 Da (TA-1) of the serum graph hasdifferent height before and after treatment.

FIG. 7 is a graph comparing values before and after MCNS treatment. InFIG. 7, the peak at 5910 Da (TA-2) has different height before and aftertreatment.

FIG. 8 is a graph comparing values before and after MCNS treatment.Samples of serum, urine and peripheral blood lymphocyte (PBNC) wereused. In FIG. 8, the peak at 5920 Da (TB-1) of the serum graph hasdifferent height before and after treatment.

FIG. 9 is a graph comparing values before and after MCNS treatment.Samples of serum, urine and peripheral blood lymphocyte (PBNC) wereused. In FIG. 9, the peaks at 10224.4 Da (TB-2) and 10793.3 Da (TB-3) ofthe serum graph have different height before and after treatment. Thepeak at 10793.3 Da (TB-3) has higher value after treatment.

FIG. 10 is a graph comparing values before and after MCNS treatment.Samples of serum, urine and peripheral blood lymphocyte (PBNC) wereused. In FIG. 10, the peaks at 13672.1 Da (TC-1) and 139801.1 Da (TC-2)of the serum graph have different height before and after treatment.These peaks have higher values after treatment. In FIG. 10, the peak at13895.6 Da (TC-3) of the urine graph has different height before andafter treatment. In FIG. 10, the peaks at 13788.5 Da (TC-4) and 13965.4Da (TC-5) of the PBNC graph have different height before and aftertreatment. These peaks have higher values after treatment.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); Cambridge Dictionary of Science and Technology(Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger el al.(eds.), Springer Verlag (1991); and Hale & Marham, The Harper CollinsDictionary of Biology (1991). As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

“Marker” in the context of the present invention refers to an organicbiomolecule, e.g., a polypeptide, which is differentially present in asample taken from patients having MCNS as compared to a comparablesample taken from subjects who do not have MCNS (e.g., MN, FGS, IgAnephropathy, or MPGN patients or healthy subjects). For examples, amarker can be a polypeptide (having a particular apparent molecularweight) which is present at an elevated level in samples of MCNSpatients compared to samples of MN, FGS, IgA nephropathy, or MPGNpatients. In another examples, a marker can be a polypeptide (having aparticular apparent molecular weight) which is present at an elevatedlevel in samples of MN, FGS, IgA nephropathy, or MPGN patients comparedto samples of MCNS patients.

A protein marker is resolved with confidence of about 0.5% variation bygas phase ion spectrometry. Thus, the term “about” in the context of amolecular weight of a marker as measured by mass spectrometry refers to0.5% variation of the marker molecular weight. For example, the markerwith an apparent molecular weight of “about 2776 Da” as measured by massspectrometry has the apparent molecular weight range of 2776±14 Da; themarker with an apparent molecular weight of “about 4423 Da” as measuredby mass spectrometry has the apparent molecular weight range of 4423±22Da; and so on.

The phrase “differentially present” refers to differences in thequantity and/or frequency of a polypeptide (of a particular apparentmolecular weight) present in a sample taken from patients having MCNS ascompared to a comparable sample taken from patients who do not have MCNS(e.g., have MN, FGS, IgA nephropathy, or MPGN). For example, a markercan be a polypeptide which is present at an elevated level or at adecreased level in samples of MCNS patients compared to samples ofsubjects who do not have MCNS. Alternatively, a marker call be apolypeptide which is detected at a higher frequency or at a lowerfrequency in samples of MCNS patients compared to samples of subjectswho do not have MCNS. A marker can be differentially present in terms ofquantity, frequency or both.

A polypeptide is differentially present between the two sets of samplesif the frequency of detecting the polypeptide in the MCNS patients'samples is statistically significantly higher or lower than in thecontrol samples. In another example, a polypeptide is differentiallypresent between the two sets of samples if it is detected at least about120%, at least about 130%, at least about 150%, at least about 180%, atleast about 200%, at least about 300%, at least about 500%, at leastabout 700%, at least about 900%, or at least about 1000% more frequentlyor less frequently observed in one set of samples than the other set ofsamples.

Alternatively or additionally, a polypeptide is differentially presentbetween the two samples if the amount of the polypeptide in one sampleis statistically significantly different from the amount of thepolypeptide in the other sample. For example, a polypeptide isdifferentially present between the two samples if it is present in onesample at least about 120%, at least about 130%, at least about 150%, atleast about 180%, at least about 200%, at least about 300%, at leastabout 500%, at least about 700%, at least about 900%, or at least about1000% greater than it is present in the other sample, or if it isdetectable in one sample and not detectable in the other.

“Diagnostic” means identifying the presence or nature of a pathologiccondition. Diagnostic methods differ in their sensitivity andspecificity. The “sensitivity” of a diagnostic assay is the percentageof diseased individuals who test positive (percent of “true positives”).Diseased individuals not detected by the assay are “false negatives.”Subjects who are not diseased and who test negative in the assay, aretermed “true negatives.” The “specificity” of a diagnostic assay is 1minus the false positive rate, where the “false positive” rate isdefined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis.

A “test amount” of a marker refers to an amount of a marker present in asample being tested. A test amount can be either in absolute amount(e.g., pg/ml) or a relative amount (e.g., relative intensity ofsignals).

A “diagnostic amount” of a marker in the context of the presentinvention refers to an amount of a marker in a subject's sample that isconsistent with a diagnosis of MCNS. A diagnostic amount can be eitherin absolute amount (e.g., μg/ml) or a relative amount (e.g., relativeintensity of signals).

A “control amount” of a marker can be any amount or a range of amountwhich is to be compared against a test amount of a marker. For example,a control amount of a marker can be the amount of a marker in a MCNSpatient, a MN, FGS, IgA nephropathy, or MPGN patient or a person withoutMCNS or MN, FGS, IgA nephropathy, or MPGN. A control amount can beeither in absolute amount (e.g., μg/ml) or a relative amount (e.g.,relative intensity of signals).

“Solid support” refers to any insoluble surface including beads orplastic strips.

“MS probe” refers to a device that, when positionally engaged in aninterrogatable relationship to an ionization source, e.g., a laserdesorption/ionization source, and in concurrent communication atatmospheric or subatmospheric pressure with the detector of thepreferred Laser Desorption/Ionization Time-Of-Flight spectrometer, canbe used to introduce ions derived from an analyte into the spectrometer.Preferred laser sources include nitrogen lasers, Nd-Yag lasers and otherpulsed laser sources. As used herein, the “MS probe” is typicallyreversibly engageable (e.g., removably insertable) with a probeinterface that positions the MS probe in an interrogatable relationshipwith the ionization source and in communication with the detector.

“SELDI MS probe” refers to an MS probe comprising an adsorbent surface.

“Adsorbent” or “adsorbent surface” refers to any material capable ofbinding an analyte (e.g., a target polypeptide). For example, a surfacefeature on a SELDI MS probe solid support or substratum can comprise anadsorbent attached thereto characterized by various adsorbent speciesthat can each be categorized as either chromatographic or biospecific,depending upon the nature of the binding interaction. Chromatographicadsorbents include ion exchange materials, metal chelators, hydrophobicinteraction adsorbents, hydrophilic interaction adsorbents, or the like.“Biospecific adsorbents,” include affinity adsorbents such aspolypeptides, enzymes, receptors, antibodies (e.g., poly- or mono-clonalantibodies, etc.), or the like, and typically have higher specificityfor a target analyte than a “chromatographic adsorbent”. Examples ofadsorbents for use in SELDI are also described in the U.S. Pat. No.6,225,047 (Hutchens and Yip, “Use of retentate chromatography togenerate difference maps,” May 1, 2001)

The term “affinity molecule” refers to a molecular substance capable ofbinding other molecules either specifically or nonspecifically.

“Wash solution” refers to solutions that are applied to a sample boundto a solid support to selectively remove sample fractions. Washsolutions are typically buffered to maintain constant pH, and/or metalion concentration, etc., but do not have to be.

“Gas phase ion spectrometer” refers to an apparatus that detects gasphase ions. In the context of this invention, gas phase ionspectrometers include an ionization source used to generate the gasphase ions. Gas phase ion spectrometers include, for example, massspectrometers, ion mobility spectrometers, and total ion currentmeasuring devices.

“Gas phase ion spectrometry” refers to a method that includes employingan ionization source to generate gas phase ions from an analytepresented on a sample presenting surface of a probe and detecting thegas phase ions with a gas phase ion spectrometer.

“Ionization source” refers to a device that directs ionizing energy to asample presenting surface of a probe to desorb and ionize analytes fromthe probe surface into the gas phase. The preferred ionization source isa laser (used in laser desorption/ionization), in particular, nitrogenlasers, Nd-Yag lasers and other pulsed laser sources. Other ionizationsources include fast atoms (used in fast atom bombardment), plasmaenergy (used in plasma desorption) and primary ions generating secondaryions (used in secondary ion mass spectrometry).

“Mass spectrometer” refers to a gas phase ion spectrometer that measuresa parameter which can be translated into mass-to-charge ratios of gasphase ions. Mass spectrometers generally include an inlet system, anionization source, an ion optic assembly, a mass analyzer, and adetector. Examples of mass spectrometers are time-of-flight, magneticsector, quadrapole filter, ion trap, ion cyclotron resonance,electrostatic sector analyzer and hybrids of these.

“Laser desorption mass spectrometer” refers to a mass spectrometer whichuses laser as a means to desorb, volatilize, and ionize an analyte.

“Fluence” refers to the energy delivered per unit area of interrogatedimage.

“Desorption/ionization” refers to generating ions by desorbing them froma solid or liquid sample with a high-energy particle beam (e.g., alaser). Desorption ionization encompasses various techniques including,e.g., surface enhanced laser desorption, matrix-assisted laserdesorption, fast atom bombardment, plasma desorption, or the like.

“Surface-enhanced laser desorption/ionization” or “SELDI” is a method ofgas phase ion spectrometry in which the surface of substrate whichpresents the analyte to the energy source plays an active role in thedesorption and ionization process. The SELDI technology is described in,e.g., U.S. Pat. No. 5,719,060 (Hutchens and Yip) and U.S. Pat. No.6,225,047 (Hutchens and Yip).

“Adsorb” refers to the detectable binding between an absorbent and amarker either before or after washing with an eluant (selectivitythreshold modifier) or a washing solution.

“Resolve,” “resolution,” or “resolution of marker” refers to thedetection of at least one marker in a sample. Resolution includes thedetection of a plurality of markers in a sample by separation andsubsequent differential detection. Resolution does not require thecomplete separation of a marker from all other markers in a mixture.Rather, any separation that allows the distinction between at least twomarkers suffices.

“Detect” refers to identifying the presence, absence or amount of theobject to be detected.

“Retention” refers to an adsorption of a marker or by an adsorbent afterwashing with an eluant or a washing solution.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers.Polypeptides can be modified, e.g., by the addition of carbohydrateresidues to form glycoproteins. The terms “polypeptide,” “peptide” and“protein” include glycoproteins, as well as non-glycoproteins.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an carbon that is bound to ahydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthee letter symbols or by the one-letter symbols recommended by theIUPAC-TUB Biochemical Nomenclature Commission.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include 32P, 35S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavidin, digoxigenin, haptens and proteins for whichantisera or monoclonal antibodies are available, or nucleic acidmolecules with a sequence complementary to a target. The detectablemoiety often generates a measurable signal, such as a radioactive,chromogenic, or fluorescent signal, that can be used to quantitate theamount of bound detectable moiety in a sample. The detectable moiety canbe incorporated in or attached to a primer or probe either covalently,or through ionic, van der Waals or hydrogen bonds, e.g., incorporationof radioactive nucleotides, or biotinylated nucleotides that arerecognized by streptavidin. The detectable moiety may be directly orindirectly detectable. Indirect detection can involve the binding of asecond directly or indirectly detectable moiety to the detectablemoiety. For example, the detectable moiety can be the ligand of abinding partner, such as biotin, which is a binding partner forstreptavidin, or a nucleotide sequence, which is the binding partner fora complementary sequence, to which it can specifically hybridize. Thebinding partner may itself be directly detectable, for example, anantibody may be itself labeled with a fluorescent molecule. The bindingpartner also may be indirectly detectable, for example, a nucleic acidhaving a complementary nucleotide sequence can be a part of a branchedDNA molecule that is in turn detectable through hybridization with otherlabeled nucleic acid molecules. (See, e.g. P. D. Fahrlander and A.Klausner, Bio/Technology 6:1165 (1988)). Quantization of the signal isachieved by, e.g., scintillation counting, densitometry, or nowcytometry.

“Antibody” refers to a polypeptide ligand substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an epitope (e.g., an antigen). Therecognized immunoglobulin genes include the kappa and lambda light chainconstant region genes, the alpha, gamma, delta, epsilon and mu heavychain constant region genes, and the myriad immunoglobulin variableregion genes. Antibodies exist, e.g., as intact immunoglobulins or as anumber of well characterized fragments produced by digestion withvarious peptidases. The includes, e.g., Fab′ and F(ab)′2 fragments. Theterm “antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It also includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies, humanizedantibodies, or single chain antibodies. “Fc” portion of an antibodyrefers to that portion of an immunoglobulin heavy chain that comprisesone or more heavy chain constant region domains, CH1, CH2 and CH3, butdoes not include the heavy chain variable region.

Methods for preparing antibodies are well-known in the art. See, e.g.,Coligan, Current Protocols in Immunology (1991); Harlow & Lane,Antibodies: A Laboratory Manual (1988); Goding, Monoclonal Antibodies:Principles and Practice (2d ed. 1986); and Kohler & Milstein, Nature256:495-497 (1975). Such techniques include, but are not limited to,antibody preparation by selection of antibodies from libraries ofrecombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

“Immunoassay” is an assay that uses an antibody to specifically bind anantigen. The immunoassay is characterized by the use of specific bindingproperties of a particular antibody to isolate, target, and/or quantifythe antigen.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. A variety of immunoassay formatsmay be used to select antibodies specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select antibodies specifically immunoreactive with aprotein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual(1988), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity). Typically a specific orselective reaction will be at least twice background signal or noise andmore typically more than 10 to 100 times background.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery of diagnostic markersthat are differentially present in the samples of MCNS patients andsubjects who do not have MCNS (e.g., have MN, FGS, IgA nephropathy, orMPGN), and the application of this discovery in the methods and kits foraiding a MCNS diagnosis. The present invention is also based upon thediscovery that the amount of therapeutic markers measured in samplestaken from a patient before and after treatment may change due to thetreatment. By monitoring the amount of one or more diagnostic ortherapeutic markers in a sample taken from a subject, the methods andkits of the invention can determine the subject's pathological status.The methods and kits of the invention can also be used in addition toconventional MCNS testing methods to confirm the presence or absence ofMCNS. The methods of the invention can be performed in a short amount oftime using minute quantities of easily obtained biological samples suchas blood, serum, urine, semen, seminal fluid, or tissue extracts.

The markers of the present invention may have any suitablecharacteristics, including any apparent molecular weights. For example,some suitable markers are present at an elevated level in samples ofMCNS patients compared to samples of MN, FGS, IgA nephropathy, or MPGNpatients. These markers may be found in a number of biological samples,and markers found in serum are preferably monitored in the methods andkits of the invention.

Each of the markers can have particular binding characteristics whichallow these markers to be enriched and measured in a sample taken from asubject under selectivity conditions that favor binding of thesemarkers.

I. Methods for Detecting Markers Using Gas Phase Ion Spectrometry

In one aspect, the invention provides methods for detecting markerswhich are differentially present in samples of a MCNS patient and aperson who does not have MCNS (e.g., MN, FGS, IgA nephropathy, or MPGNpatient). Any one or combination of markers described are within thescope of this aspect of this invention and can be detected. The methodsfor detecting these markers have many applications. For example, onemarker or combination of markers can be measured to differentiatebetween MCNS and MN, FGS, IgA nephropathy, or MPGN, and thus are usefulas an aid in the diagnosis of MCNS in a patient. In another example, thepresent methods for detecting these markers can be applied to in vitroMCNS cells or in vivo animal models for MCNS to assay for and identifycompounds that modulate expression of these markers.

In another aspect, the invention provides for methods for evaluating theprogress of the disease and the therapeutic value of agents used totreat kidney disease by measuring the amount of therapeutic markers insamples taken from a patient before and after treatment. The amount ofthe markers may change due to the treatment. Therapeutic markers aredifferentially present in a sample obtained from a patient before andafter treatment.

A. Gas Phase Ion Spectrometry Detection

In one embodiment of the detection method, a gas phase ion spectrometercan be used. This method comprises: (a) contacting a sample with asubstrate comprising an adsorbent thereon under conditions to allowbinding between a marker and the adsorbent; and (b) detecting the markerbound to the adsorbent by gas phase ion spectrometry.

The detection of these markers can be enhanced using certain selectivityconditions (e.g., types of adsorbents used or washing solutions). In apreferred embodiment, the same or substantially the same selectivityconditions that were used to discover the markers can be used in themethods for detecting a marker in a sample. For example, a substratecomprising an adsorbent having a hydrophobic group and an anionic group(e.g., polystyrene latex beads functionalized with a sulfonate group)can be used. In another example, a substrate comprising an adsorbenthaving a hydrophobic group (e.g., an aliphatic C16 hydrocarbon group)can be used. In yet another example, a substrate comprising an adsorbenthaving a metal ion bound to a metal chelating group (e.g., nickel metalions chelated by nitrilotriacetic acid groups) as adsorbents can beused. In some embodiments, an adsorbent can be antibodies thatspecifically bind to the markers. Preferably, a sample is serum takenfrom a subject.

In one embodiment, a substrate comprising an adsorbent can be in theform of a probe, which is removably insertable into a gas phase ionspectrometer. For example, a substrate can be in the form of a stripwith adsorbents on its surface. In another embodiment, a substratecomprising an adsorbent can be positioned onto another substrate to forma probe, which is removably insertable into a gas phase ionspectrometer. For example, a substrate comprising an adsorbent can be asolid phase, such as a polymeric or glass bead with a functional groupfor binding a marker, which can be subsequently positioned on a secondsubstrate to form a probe. For example, the second substrate can be inthe form of a strip, or a plate having a series of wells at apredetermined addressable locations. One advantage of this embodiment isthat the marker can be adsorbed to the first substrate in one physicalcontext, and transferred to the second substrate, which can then besubmitted for analysis by gas phase ion spectrometry. The probe can bein any shape as long as it is removably insertable into a gas phase ionspectrometer.

The probe can also be adapted for use with inlet systems and detectorsof a gas phase ion spectrometer. For example, the probe can be adaptedfor mounting in a horizontally and/or vertically translatable carriagethat horizontally and/or vertically moves the probe to a successiveposition without requiring repositioning of the probe by hand.

The probe substrate is preferably made of a material that is capable ofsupporting adsorbents. For example, the probe substrate material caninclude, but is not limited to, insulating materials (e.g., glass,ceramic), semi-insulating materials (e.g., silicon wafers), orelectrically conducting materials (e.g., metals, such as nickel, brass,steel, aluminum, gold, or electrically conductive polymers), organicpolymers, biopolymers, or any combinations thereof.

The probe substrate surface can be conditioned to bind markers. Forexample, in one embodiment, the surface of the probe substrate can beconditioned (e.g., chemically or mechanically such as roughening) toplace adsorbents on the surface. The adsorbent comprises functionalgroups for binding with a marker. In some embodiments, the substratematerial itself can also contribute to adsorbent properties and may beconsidered part of an “adsorbent.”

Any number of different adsorbents can be used as long as they havebinding characteristics suitable for binding the markers of the presentinvention. The adsorbents can comprise a hydrophobic group, ahydrophilic group, a cationic group, an anionic group, a metal ionchelating group, or antibodies which specifically bind to antigens, or acombination thereof (sometimes referred to as “a mixed mode” adsorbent).Exemplary adsorbents comprising a hydrophobic group include matriceshaving aliphatic hydrocarbons, e.g., C1-C18 a aliphatic hydrocarbons andmatrices having aromatic hydrocarbon functional group such as phenylgroups. Exemplary adsorbents comprising a hydrophilic group includesilicon oxide (i.e., glass), or hydrophilic polymers such aspolyethylene glycol, dextran, agarose, or cellulose. Exemplaryadsorbents comprising a cationic group include matrices of secondary,tertiary or quaternary amines. Exemplary adsorbents comprising ananionic group include matrices of sulfate anions (SO3-) and matrices ofcarboxylate anions (i.e., COO-) or phosphate anions (OPO3-). Exemplaryadsorbents comprising metal chelating groups include organic moleculesthat have one or more electron donor groups which form coordinatecovalent bonds with metal ions, such as copper, nickel, cobalt, zinc,iron, and other metal ions such as aluminum and calcium. Exemplaryadsorbents comprising an antibody include antibodies that are specificfor any one of the markers provided herein. In preferred embodiments,adsorbents are substantially similar to or the same as the adsorbentswhich were used to enrich and identify the markers.

Adsorbents can be placed on the probe substrate in continuous ordiscontinuous patterns. If continuous, one or more adsorbents can beplaced on the substrate surface. If multiple types of adsorbents areused, the substrate surface can be coated such that one or more bindingcharacteristics vary in one or two-dimensional gradient. Ifdiscontinuous, plural adsorbents can be placed in predeterminedaddressable locations on the substrate surface. The addressablelocations can be arranged in any pattern, but are preferably in regularpattern, such as lines, orthogonal arrays, or regular curves (e.g.,circles). Each addressable location may comprise the same or differentadsorbent.

The probes can be produced using any suitable methods depending on theselection of substrate materials and/or adsorbents. For example, thesurface of a metal substrate can be coated with a material that allowsderivitization of the metal surface. More specifically, a metal surfacecan be coated with silicon oxide, titanium oxide or gold. Then surfacecan be derivatized with a bifunctional linker, one end of which cancovalently bind with a functional group on the surface and the other endof which can be further derivatized with groups that function as anadsorbent. In another example, a porous silicon surface generated fromcrystalline silicon can be chemically modified to include adsorbents forbinding markers. In yet another example, adsorbents with a hydrogelbackbone can be formed directly on the substrate surface by in situpolymerizing a monomer solution which comprises, e.g., substitutedacrylamide monomers, substituted acrylate monomers, or derivativesthereof comprising a functional group of choice as an adsorbent.

Probes suitable for use in the invention are also described in, e.g.,WO98/59361, U.S. Ser. No. 60/131,652, filed Apr. 29, 1999, and Wei etal., Nature 399:243-30 246 (1999).

The probe substrate comprising an adsorbent contacts a sample. Thesample is preferably a biological fluid sample. Examples of biologicalfluid samples include blood, serum, urine, semen, seminal fluid ortissue extracts. In a preferred embodiment, the biological fluidcomprises serum.

The sample can be solubilized in or admixed with an eluant. The probesubstrate comprising an adsorbent then contacts the solution using anytechniques including bathing, soaking, dipping, spraying, washing over,or pipetting, etc. Generally, a volume of sample containing from a fewattomoles to 100 picomoles of marker in about 1 μl to 500 μl issufficient for binding to the adsorbent.

The sample can contact the probe substrate comprising an adsorbent for aperiod of time sufficient to allow the marker to bind to the adsorbent.Typically, the sample and the substrate comprising the adsorbent arecontacted for a period of between about 30 seconds and about 12 hours,and preferably, between about 30 seconds and about 15 minutes.

The temperature at which the sample contacts the probe substratecomprising an adsorbent can be a function of the particular sample andthe selected probe. Typically, the sample is contacted to the probesubstrate under ambient temperature and pressure conditions. For somesamples, however, modified temperature (typically 4° C. through 37° C.),and pressure conditions can be desirable, which conditions aredeterminable by those skilled in the art.

After the probe substrate comprising an adsorbent contacts the sample orsample solution, it is preferred that unbound materials on the probesubstrate surface are washed out so that only the bound materials remainon the substrate surface. Washing a probe substrate surface can beaccomplished by, e.g., bathing, soaking, dipping, rinsing, spraying, orwashing the substrate surface with an eluant or a washing solution. Amicrofluidics process is preferably used when a washing solution such asan eluant is introduced to small spots of adsorbents on the probe.Typically, the washing solution can be at a temperature of between 0° C.and 100° C., preferably between 4° C. and 37° C.

Any suitable washing solutions or eluants can be used to wash the probesubstrate surface. For example, organic solutions or aqueous solutionscan be used. Preferably, an aqueous solution is used. Exemplary aqueoussolutions include a HEPES buffer, a Tris buffer, a phosphate bufferedsaline, etc. The selection of a particular washing solution or an eluantis dependent on other experimental conditions (e.g., types of adsorbentsused or markers to be detected), and can be determined by those of skillin the art. For example, if a probe comprising a hydrophobic group and asulfonate group as adsorbents is used, then an aqueous solution, such asa HEPES buffer, may be preferred. In another example, if a probecomprising a metal binding group as an adsorbent is used. then anaqueous solution, such as a phosphate buffered saline, may be preferred.In yet another example, if a probe comprising a hydrophobic group isused, then water may be preferred as a washing solution.

Optionally, an energy absorbing molecule (e.g., in solution) can beapplied to markers or other substances bound on the probe substratesurface. Spraying, pipetting, or dipping can be used. This can be doneafter unbound materials are washed off of the probe substrate surface.An energy absorbing molecule refers to a molecule that absorbs energyfrom an energy source in a gas phase ion spectrometer, thereby assistingdesorption of markers or other substances from a probe surface.Exemplary energy absorbing molecules include cinnamic acid derivatives,sinapinic acid and dihydroxybenzoic acid.

After the marker is bound to the probe, it is detected using gas phaseion spectrometry. Markers or other substances bound to the adsorbents onthe probes can be analyzed using a gas phase ion spectrometer. Thisincludes, e.g., mass spectrometers, ion mobility spectrometers, or totalion current measuring devices. The quantity and characteristics of themarker can be determined using gas phase ion spectrometry. Othersubstances in addition to the marker of interest can also be detected bygas phase ion spectrometry.

In one embodiment, a mass spectrometer can be used to detect markers onthe probe. In a typical mass spectrometer, a probe with a marker isintroduced into an inlet system of the mass spectrometer. The marker isthen ionized by an ionization source such as a laser, fast atombombardment, or plasma. The generated ions are collected by an ion opticassembly, and then a mass analyzer disperses and analyzes the passingions. The ions exiting the mass analyzer are detected by a detector. Thedetector then translates information of the detected ions intomass-to-charge ratios. Detection of the presence of a marker or othersubstances will typically involve detection of signal intensity. This,in turn, can reflect the quantity and character of a marker bound to theprobe.

In a preferred embodiment, a laser desorption time-of-flight massspectrometer is used with the probe of the present invention. In laserdesorption mass spectrometry, a probe with a bound marker is introducedinto an inlet system. The marker is desorbed and ionized into the gasphase by laser from the ionization source. The ions generated arecollected by an ion optic assembly, and then in a time-of-flight massanalyzer, ions are accelerated through a short high voltage field andlet drift into a high vacuum chamber. At the far end of the high vacuumchamber, the accelerated ions strike a sensitive detector surface at adifferent time. Since the time-of-flight is a function of the mass ofthe ions, the elapsed time between ionization and impact can be used toidentify the presence or absence of molecules of specific mass. As anyperson skilled in the art understands, any of these components of thelaser desorption time-of-flight mass spectrometer can be combined withother components described herein in the assembly of mass spectrometerthat employs various means of desorption, acceleration, detection,measurement of time, etc.

In another embodiment, an ion mobility spectrometer can be used todetect and characterize a marker. The principle of ion mobilityspectrometry is based on different mobility of ions. Specifically, ionsof a sample produced by ionization move at different rates, due to theirdifference in, e.g., mass, charge, or shape, through a tube under theinfluence of an electric field. The ions (typically in the form of acurrent) are registered at the detector which can then be used toidentify a marker or other substances in the sample. One advantage ofion mobility spectrometry is that it can operate at atmosphericpressure.

In yet another embodiment, a total ion current measuring device can beused to detect and characterize markers. This device can be used whenthe probe has a surface chemistry that allows only-a single type ofmarker to be bound. When a single type of marker is bound on the probe,the total current generated from the ionized marker reflects the natureof the marker. The total ion current produced by the marker can then becompared to stored total ion current of known compounds. Characteristicsof the marker can then be determined.

Data generated by desorption and detection of markers can be analyzedwith the use of a programmable digital computer. The computer programgenerally contains a readable medium that stores codes. Certain code canbe devoted to memory that includes the location of each feature on aprobe, the identity of the adsorbent at that feature and the elutionconditions used to wash the adsorbent. Using this information, theprogram can then identify the set of features on the probe definingcertain selectivity characteristics (e.g., types of adsorbent andeluants used). The computer also contains code that receives as input,data on the strength of the signal at various molecular masses receivedfrom a particular addressable location on the probe. This data canindicate the number of markers detected, optionally including thestrength of the signal and the determined molecular mass for each markerdetected.

Data analysis can include the steps of determining signal strength(e.g., height of peaks) of a marker detected and removing “outerliers”(data deviating from a predetermined statistical distribution). Forexample, the observed peaks can be normalized, a process whereby theheight of each peak relative to some reference is calculated. Forexample, a reference can be background noise generated by instrument andchemicals (e.g., energy absorbing molecule) which is set as zero in thescale. Then the signal strength detected for each marker or othersubstances can be displayed in the form of relative intensities in thescale desired (e.g., 100). Alternatively, a standard may be admittedwith the sample so that a peak from the standard can be used as areference to calculate relative intensities of the signals observed foreach marker or other markers detected.

The computer can transform the resulting data into various formats fordisplaying. In one format, referred to as “spectrum view or retentatemap,” a standard spectral view can be displayed, wherein the viewdepicts the quantity of marker reaching the detector at each particularmolecular weight. In another format, referred to as “peak map,” only thepeak height and mass information are retained from the spectrum view,yielding a cleaner image and enabling markers with nearly identicalmolecular weights to be more easily seen. In yet another format,referred to as “gel view,” each mass from the peak view can be convertedinto a grayscale image based on the height of each peak, resulting in anappearance similar to bands on electrophoresis gels. In yet anotherformat, referred to as “3-D overlays,” several spectra can be overlayedto study subtle changes in relative peak heights. In yet another format,referred to as “difference map view,” two or more spectra can becompared, conveniently highlighting unique markers and markers which areup- or down-regulated between samples. Marker profiles (spectra) fromany two samples may be compared visually.

Using any of the above display formats, it can be readily determinedfrom the signal display whether a diagnostic marker having a particularmolecular weight (e.g., about 2955.3 Da (DA-1), 6116.6 Da (DA-2), 5910.0Da (DA-3), 2962.5 Da (DB-1), 6130.8 Da (DB-2), or 3161.5 Da (DB-3)) isdetected from a sample. Alternatively, it can be readily determined fromthe signal display whether a therapeutic marker having an apparentmolecular weight of about 2952.3 Da (TA-1), 5910.0 Da (TA-2), 5922.6 Da(TB-1), 10224.4 Da (TB-2), 10793.3 Da (TB-3), 13672.1 Da (CA-1), 13980.1Da (CA-2), 13895.6 Da (CA-3), 13788.5 Da (CA-4), or 13965.4 Da (CA-5) isdetected from a sample. Moreover, from the strength of signals, theamount of diagnostic or therapeutic markers bound on the probe surfacecan be determined.

B. Immunoassay Detection

In another embodiment of the detection method, an immunoassay can beused to qualitatively or quantitatively detect and analyze diagnostic ortherapeutic markers in a sample. This method comprises: (a) providing anantibody that specifically binds to a marker, wherein the marker is apolypeptide which is differentially present in samples of a MCNS patientand a subject who does not have MCNS (e.g., MN, FGS, IgA nephropathy, orMPGN patients) or is differentially present in a sample obtained from apatient before and after treatment; (b) contacting a sample with theantibody, and (c) detecting the presence of a complex of the antibodybound to the marker in the sample.

To prepare an antibody that specifically binds to a marker, purifiedmarkers or their nucleic acid sequences can be used. Nucleic acid andamino acid sequences for other markers can be obtained by furthercharacterization of these markers. For example, each marker can bepeptide mapped with a number of enzymes (e.g., trypsin, V8 protease,etc.). The molecular weights of digestion fragments from each marker canbe used to search the databases, such as SwissProt database, forsequences that will match the molecular weights of digestion fragmentsgenerated by various enzymes. Using this method, the nucleic acid andamino acid sequences of other markers can be identified if these markersare known proteins in the databases.

Alternatively, the proteins can be sequenced using protein laddersequencing. Protein ladders can be generated by, for example,fragmenting the molecules and subjecting fragments to enzymaticdigestion or other methods that sequentially remove a single amino acidfrom the end of the fragment. Methods of preparing protein ladders aredescribed, for example, in International Publication WO 93/24834 (Chaitet al.) and U.S. Pat. No. 5,792,664 (Chait et al.). The ladder is thenanalyzed by mass spectrometry. The difference in the masses of theladder fragments identify the amino acid removed from the end of themolecule.

If the markers are not known proteins in the databases, nucleic acid andamino acid sequences can be determined with knowledge of even a portionof the amino acid sequence of the marker. For example, degenerate probescan be made based on the N-terminal amino acid sequence of the marker.These probes can then be used to screen a genomic or cDNA librarycreated from a sample from which a marker was initially detected. Thepositive clones can be identified, amplified, and their recombinant DNAsequences can be subcloned using techniques which are well known. See,e.g., Current Protocols for Molecular Biology (Ausbel et al., GreenPublishing Assoc. and Wiley-Interscience 1989) and Molecular Cloning: ALaboratory Manual, 2nd Ed. (Sambrook el al., Cold Spring HarborLaboratory, New York 1989).

Using the purified markers or their nucleic acid sequences, antibodiesthat specifically bind to a marker can be prepared using any suitablemethods known in the art. See, e.g., Coligan, Current Protocols inImmunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual(1988); Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Milstein, Nature 256:495-497 (1975). Such techniquesinclude, but are not limited to, antibody preparation by selection ofantibodies from libraries of recombinant antibodies in phage or similarvectors, as well as preparation of polyclonal and monoclonal antibodiesby immunizing rabbits or mice (see, e.g., Huse et al., Science246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989)).

After the antibody is provided, a marker can be detected and/orquantified using any of a number of well recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,2411 4,376,110;4,517,288; and 4,837,168). Useful assays include, for example, an enzymeimmune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), aradioimmune assay (NA), a Western blot assay, or a slot blot assay. Fora review of the general immunoassays, see also, Methods in Cell Biology:Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic andClinical Immunology (Stites & Terr, eds., 7th ed. 1991); Harlow & Lane,Antibodies, A Laboratory Manual (1988).

Generally, a sample obtained from a subject can be contacted with theantibody that specifically binds the marker. Optionally, the antibodycan be fixed to a solid support to facilitate washing and subsequentisolation of the complex, prior to contacting the antibody with asample. Examples of solid supports include glass or plastic in the formof, e.g., a microtiter plate, a stick, a bead, or a microbead.Antibodies can also be attached to a probe substrate or ProteinChip®array described above. The sample is preferably a biological fluidsample taken from a subject. Examples of biological fluid samplesinclude blood, serum, urine, semen, seminal fluid or tissue extracts.The sample can be diluted with a suitable eluant before contacting thesample to the antibody.

After incubating the sample with antibodies, the mixture is washed andthe antibody-marker complex formed can be detected. This can beaccomplished by incubating the washed mixture with a detection reagent.This detection reagent may be, e.g., a second antibody which is labeledwith a detectable label. Exemplary detectable labels include magneticbeads (e.g., DYNABEADS™), fluorescent dyes, radiolabels, enzymes (e.g.,horse radish peroxide, alkaline phosphatase and others commonly used inan ELISA), and colorimetric labels such as colloidal gold or coloredglass or plastic beads. Alternatively, the marker in the sample can bedetected using an indirect assay, wherein, for example, a second,labeled antibody is used to detect bound marker-specific antibody,and/or in a competition or inhibition assay wherein, for example, amonoclonal antibody which binds to a distinct epitope of the marker areincubated simultaneously with the mixture.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,marker, volume of solution, concentrations and the like. Usually theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Immunoassays can be used to determine the presence or absence of amarker in a sample as well as the quantity of a marker in a sample.First, a test amount of a marker in a sample can be detected using theimmunoassay methods described above. If a marker is present in thesample, it will form an antibody-marker complex with an antibody thatspecifically binds the marker under suitable incubation conditionsdescribed above. The amount of an antibody-marker complex can bedetermined by comparing to a standard. A standard can be, e.g., a knowncompound or another protein known to be present in a sample. As notedabove, the test amount of marker need not be measured in absolute units,as long as the unit of measurement can be compared to a control.

The methods for detecting these markers in a sample have manyapplications. For example, one or more markers can be measured to aidMCNS diagnosis or prognosis. For example, these diagnostic markersinclude polypeptides having an apparent molecular weight of about 2955.3Da (DA-1), 6116.6 Da (DA-2), 5910.0 Da (DA-3), 2962.5 Da (DB-1), 6130.8Da (DB-2), or 3161.5 Da (DB-3). In another example, the methods fordetection of the markers can be used to monitor responses in a subjectto MCNS therapeutic treatment For example, these therapeutic markersinclude polypeptides having an apparent molecular weight of about 2952.3Da (TA-1), 5910.0 Da (TA-2), 5922.6 Da (TB-1), 10224.4 Da (TB-2),10793.3 Da (TB-3), 13672.1 Da (CA-1), 13980.1 Da (CA-2), 13895.6 Da(CA-3), 13788.5 Da (CA-4), or 13965.4 Da (CA-5). In another example, themethods for detecting markers can be used to assay for and to identifycompounds that modulate expression of these markers in vivo or in vitro.

II. Methods for Diagnosing MCNS Using Test Amount of Markers

In another aspect, the invention provides methods for aiding a MCNSdiagnosis using a marker which is differentially present in samples of aMCNS patient and a subject who does not have MCNS (e.g., a MN, FGS, IgAnephropathy, or MPGN patient). Any one or combination of markersdescribed above can be used for aiding MCNS diagnosis. Compared to thecurrent MCNS tests available, the present methods provide a quick andsimple way to differentiate if a subject has MCNS or MN, FGS, IgAnephropathy, or MPGN, and thus aiding a MCNS diagnosis. The methodscomprise: (a) determining a test amount of a marker in a sample from thesubject; and (b) determining whether the test amount is a diagnosticamount consistent with a diagnosis of MCNS.

In step (a), a test amount of a marker in a sample from a subject isdetermined. Any suitable samples can be obtained from a subject.Preferably, a sample is a biological fluid sample taken from a subjectbeing tested. Examples of biological fluid samples include blood, serum,urine, semen, seminal fluid or tissue extracts. Moreover, testing aserum sample does not require an invasive procedure, such as a biopsy.

After a sample is obtained, any suitable method can be used to determinea test amount of the marker in a sample from a subject being tested. Forexample, gas phase ion spectrometry or an immunoassay can be used.

In one embodiment, gas phase ion spectrometry can be used to determine atest amount of a marker in a sample from a subject. First, one or moremarkers can be detected with gas phase ion spectrometry using themethods described above. After the marker is detected by a gas phase ionspectrometer, the test amount of marker can be determined. For example,a signal is displayed at the molecular weight of the marker of interest.Based on the strength or magnitude of the displayed signal, the amountof marker in a sample being tested can be determined. It is noted thatthe test amount of marker in a sample need not be measured in absoluteunits, but can be in relative units as long as it can be comparedqualitatively or quantitatively to a control amount of a marker. Forexample, as described above, the amount of the marker detected can bedisplayed in terms of relative intensity based on the background noise.Preferably, the test amount and the control amount of markers aremeasured under the same conditions.

If desired, the absolute amount of a marker can be determined bycalibration. From the peak intensity vs. concentration plot, theabsolute amount of a marker in any sample being tested can bedetermined.

In another embodiment, an immunoassay can be used to determine a testamount of a marker in a sample from a subject. First, a test amount of amarker in a sample can be detected using the immunoassay methodsdescribed above. If a marker is present in the sample, it will form allantibody-marker complex with an antibody that specifically binds themarker under suitable incubation conditions described above. The amountof an antibody-marker complex can be determined by comparing to astandard. As noted above, the test amount of marker need not be measuredin absolute units, as long as the unit of measurement can be compared toa control amount.

After a test amount of marker is determined using either method, thenbased on the test amount, it can be determined whether a subject hasMCNS. This determination can be made by any suitable methods. Forexample, the test amount can be compared to a control amount which canbe a value or a range of values determined as follows.

In one embodiment, the control amount can be an amount of a markerpresent in comparable samples from MN, FGS, IgA nephropathy, or MPGNpatients. The control amount is measured under the same or substantiallysimilar experimental conditions as in measuring the test amount. Forexample, if a test sample is obtained from a subject's serum and amarker is detected using a particular probe, then a control amount ofthe marker is preferably determined from a serum sample of a MN, FGS,IgA nephropathy, or MPGN patient using the same probe. It is preferredthat the control amount of marker is determined based upon a significantnumber of samples from subjects who do not have MCNS (e.g., MN, FGS, IgAnephropathy, or MPGN patients) so that it reflects variations of themarker amounts in that population. If the test amount of marker issignificantly increased compared to the control amount of marker whichis known to be elevated in samples of MCNS patients (e.g., 5753 Da),then it can be a positive indication that a subject being tested hasMCNS. For example, if the test amount is increased by 1.5 fold,preferably by 2 fold, more preferably by 5 fold, or most preferably by10 fold compared to the control amount, then the test amount is adiagnostic amount which is consistent with a diagnosis of MCNS. Theconverse would apply for markers that are known to be elevated in thesamples of MN, FGS, IgA nephropathy, or MPGN patients than MCNS patients(e.g., 3600 Da).

In another embodiment, a control amount can be an amount of a markerpresent in comparable samples from a MCNS patient. Again, it ispreferred that the control amount of a marker is determined based upon asignificant number of samples taken from MCNS patients so that itreflects variations of the marker amounts in that population. If thetest amount of the marker is about the same as the control amount of themarker, then it can be a positive indication that a subject being testedhas MCNS.

In yet another embodiment, a control amount can be an mount of a markerpresent in comparable samples from a normal person (i.e., who is knownto be free of MCNS and MN, FGS, IgA nephropathy, or MPGN). It ispreferred that the control amount of marker is determined based upon asignificant number of samples taken from normal persons so that itreflects variations of the marker amounts in that population. If thecontrol amount of a particular marker is significantly lower than theamount of the same marker present in comparable samples of MCNSpatients, then this marker can be used to diagnose MCNS and rule out MN,FGS, IgA nephropathy, or MPGN in a single test. In such a case, if thetest amount of marker is significantly increased compared to the controlamount of marker, then it can be a positive indication that a subjectbeing tested has MCNS. For example, if the test amount is increased by1.5 fold, preferably by 2 fold, more preferably by 5 fold, mostpreferably by 10 fold compared to the control amount, then the testamount is a diagnostic amount which is consistent with a diagnosis ofMCNS. The converse would apply for markers that are known to be elevatedin the samples of MN, FGS, IgA nephropathy, or MPGN patients than MCNSpatients.

Data generated by mass spectrometry can then be analyzed by a computersoftware. The software can comprise code that converts signal from themass spectrometer into computer readable form. The software also caninclude code that applies an algorithm to the analysis of the signal todetermine whether the signal represents a “peak” in the signalcorresponding to a marker of this invention, or other useful markers.The software also can include code that executes an algorithm thatcompares signal from a test sample to a typical signal characteristic of“normal” and MCNS and determines the closeness of fit between the twosignals. The software also can include code indicating which the testsample is closest to, thereby providing a probable diagnosis.

III. Kits

In yet another aspect, the invention provides kits for aiding adiagnosis of MCNS, wherein the kits can be used to detect the markers ofthe present invention. For example, the kits can be used to detect anyone or combination of markers described above, which markers aredifferentially present in samples of a MCNS patient and a MN, FGS, IgAnephropathy, or MPGN patient. The kits may be used to detect any one ora combination of markers, which markers are differentially present insamples obtained from a patient before and after treatment. The kits ofthe invention have many applications. For example, the kits can be usedto differentiate if a subject has MCNS or MN, FGS, IgA nephropathy, orMPGN, thus aiding a MCNS diagnosis. In another example, the kits can beused to determine if a patient is responding to treatment. In anotherexample, the kits can be used to identify compounds that modulateexpression of the markers in in vivo animal models for MCNS.

In one embodiment, a kit comprises: (a) a substrate comprising anadsorbent thereon, wherein the adsorbent is suitable for binding amarker, and (b) a washing solution or instructions for making a washingsolution, wherein the combination of the adsorbent and the washingsolution allows detection of the marker using gas phase ionspectrometry. Such kits can be prepared from the materials describedabove, and the previous discussion of these materials (e.g., probesubstrates, adsorbents, washing solutions, etc.) is fully applicable tothis section and need not be repeated.

In some embodiments, the kit may comprise a first substrate composing anadsorbent thereon (e.g., a particle functionalized with an adsorbent)and a second substrate onto which the first substrate can be positionedto form a probe which is removably insertable into a gas phase ionspectrometer. In other embodiments, the kit may comprise a singlesubstrate which is in the form of a removably insertable probe withadsorbents on the substrate.

Optionally, the kit can further comprise instructions for suitableoperational parameters in the form of a label or a separate insert. Forexample, the kit may have standard instructions informing a consumer howto wash the probe after a sample of serum is contacted on the probe.

In another embodiment, a kit comprises (a) an antibody that specificallybinds to a marker; and (b) a detection reagent. Such kits can beprepared from the materials described above, and the previous discussionregarding the materials (e.g., antibodies, detection reagents,immobilized supports, etc.) is fully applicable to this section and neednot be repeated.

In either embodiment, the kit may optionally further comprise a standardor control information so that the test sample can be compared with thecontrol information standard to determine if the test amount of a markerdetected in a sample is a diagnostic amount consistent with a diagnosisof MCNS.

EXAMPLES

The following examples are offered by way of illustration, not by way oflimitation.

A. Identification of Markers Using IMAC3-Cu ProteinChip® Array

The Immobilized Metal Affinity Capture array, called “IMAC3” arrays(Ciphergen Biosystems, Inc., Fremont, Calif. USA), can be used tocapture molecules that bind divalent cationic metals such as nickel,gallium, copper and zinc. The active spots contain nitrilotriacetic acidgroups on the surface that chelate the metal ions. Proteins applied tothe chip surface may bind to the chelated metal ion through histidine,tryptophan, cysteine, and phosphorylated amino acids.

Since IMAC3 chips are manufactured in a metal-free form, IMAC3-Cu chipswere prepared as follows. Each spot was outlined using a hydrophobic penand allowed to dry. The chip was then assembled in the bioprocessor.After loading 50 μl of 100 mM copper sulfate to each spot, the chip wasshaken for 5 minutes, the excess copper was removed with runningdeionized water, and the chip was shaken for 5 minutes with an excess of50 mM sodium acetate, pH 4. The chip was further rinsed under runningdeionized water. Binding buffer (PBS) was applied to each spot andshaken for 5 min. The surface was wiped dry around the spots and removedexcess buffer without touching the active surface. IMAC3-Cu comprising achelated copper metal ion as an adsorbent was thus prepared.

Serum samples were obtained from patients with MCNS, MN, FGS, IgAnephropathy or MPGN. 0.5 μl of each serum sample was diluted 1/10 with45 μl of PBS.

The diluted sample solution was added to a spot of adsorbent ofIMAC3-Cu. The chip was shaken for 20 min. Each spot was washed with 200μl PBS three times and the chip was removed from the bioprocessor. Afterrinsing with water and being dried, 5 μl of saturated sinapinic acid wasapplied on the spots twice as a matrix. The chip was analyzed with theProteinChip® System (Ciphergen Biosystems, Inc.).

FIGS. 1, 2, and 3 illustrate the results. As shown in FIGS. 1 and 2,proteins of apparent molecular weight of about 2955 Da (DA-1) and 6117Da (DA-2) were found to be very abundant in samples from MCNS patientsthan samples from patients of other diseases.

B. Identification of Markers Using WCX2 ProteinChip® Array

The Weak Cation Exchange array, called “WCX2” arrays can be used toanalyze molecules with a positive charge on the surface. The activespots contain weak anionic carboxylate groups that interact with thepositive charges on the surface on the analyte, e.g., lysine, arginineor histidine. The chip was assembled in the bioprocessor, and wasincubated for 5 minutes with binding buffer (200 μl of 50 mM sodiumacetate for pH4 and 50 mM sodium phosphate for pH6). The incubation wasrepeated twice.

Serum samples were prepared using substantially the same solutions andprocedures described above. The diluted sample solution was added to aspot of adsorbent of WCX2. After the chip was shaken for 20 minutes,each spot was washed with binding buffer three times, and the chip wasremoved from the bioprocessor. The spots were further rinsed with water,dried, and applied sinapinic acid as a matrix. The chip was analyzedwith the ProteinChip® System (Ciphergen Biosystems, Inc.)

FIGS. 4 and 5 illustrate the results. As shown in FIGS. 4 and 5,proteins of apparent molecular weight of about 2963 Da (DB-1), 3162 Da(DB-2) and 6131 Da (DB-3) were found to be very abundant in samples fromMCNS patients than samples from patients of other diseases.

C. Identification of Markers for Evaluating the Therapeutic Value ofAgents for Treating Kidney Disease Using IMAC3-Cu, WCX2 and SAX2ProteinChip® Array

IMAC3-Cu chip and WCX2 chip were prepared and used in substantially thesame methods described above.

The strong Anion Exchange array, called the “SAX2” chip, can be used toanalyze molecules with a negative charge on the surface. The activespots contain cationic, quaternary ammonium groups that interact withthe negative charges on the surface of target proteins, e.g., asparticacid or glutamic acid. The chip was assembled in the bioprocessor and200 μl binding buffer (50 mM Tris-HCl for pH8 and 50 mM Sodium phosphatefor pH6) was added to each well. The chip was then incubated for 5minutes at room temperature with vigorous shaking. The buffer wasremoved from the wells, the sample solution was added immediately, andthe chip was incubated with vigorous shaking for 20 minutes. Then, thesamples were removed from the wells and the wells were washed threetimes with binding buffer. The chip was removed from the bioprocessor,rinsed with water and air-dried. After applying sinapinic acid as amatrix, the chip was analyzed with the ProteinChip® System (CiphergenBiosystems, Inc.)

Serum, urine and PBNC samples were obtained from a patient with MCNSbefore and after the treatment. Serum was diluted to 1/10, urine to ⅕,and PBNC to 1.2 mg/ml.

FIGS. 6 and 7 shows the results using IMAC3-Cu chips. In serum samplesolution, a protein of about 2952 Da (TA-1) and 5910 Da (TA-2)disappeared after the treatment (FIGS. 6 and 7).

FIGS. 8 and 9 illustrate the results using WCX2 chips. When using pH6buffer, proteins of apparent molecular weight of about 5923 Da (TB-1)and 10224 Da (TB-2) in serum sample vanished after the treatment (FIGS.8 and 9) and a protein of about 10793 Da (TB-3) appeared to thecontrary.

FIG. 10 illustrates the results using SAX2 chips. As shown in FIG. 10,the ratio of a protein of apparent molecular weight of about 13980 Da(TC-2) to a protein of about 13672 Da (TC-1) in serum sample solutionwas reversed after the treatment. As shown in FIG. 10, a protein ofapparent molecular weight of about 13896 Da (TC-3) in urine samplevanished after the treatment. When using pH8 buffer, the ratio of aprotein of about 13965 Da (TC-5) to a protein of 13789 Da (TC-4) in PBNCsample was reversed after the treatment (FIG. 10).

D. Identification of Markers by Protein Ladder Sequencing

One marker, the 5910 Da marker (DA-3 and TA-2) was identified by proteinladder sequencing. Serum proteins containing the marker were separatedon polyacrylamide gels, electroblotted onto Immobilon-PSQ PVDF membrane(SIGMA), the band corresponding to the 5910 Da marker was isolated andthe N-Terminal amino acid sequence was identified. A comparison of theN-Terminal sequence with the publicly available databases indicated thatthe 5910 Da marker was the α-2-HS glycoprotein β chain (mature form)(see Haglund et al., Biochemical Journal 357:437-445 (2001)). A minorcomponent also was found in the band and identified to be humanapolipoprotein AII. Polyclonal and monoclonal antibodies directedagainst the 5910 Da marker may be made as described in section I. B. byimmunizing rabbits or mice with the whole 5910 Da marker or withpeptides derived from the marker. These new antibodies, or existingpolyclonal or monoclonal antibodies may be used in the immunoassaysdescribed in section I. B. to quantitatively and qualitatively detectand analyze the marker in diagnostic and treatment samples. Polyclonaland monoclonal antibodies directed against the 5910 Da marker may bebound to a substrate and used as a specific adsorbent for gas phase ionspectrometry detection described in section I. A. to quantitatively andqualitatively detect and analyze the marker in diagnostic and treatmentsamples.

The present invention provides novel materials and methods for aidingMCNS diagnosis using markers that are differentially present in samplesof a MCNS patient and a subject who does not have MCNS. While specificexamples have been provided, the above description is illustrative andnot restrictive. Any one or more of the features of the previouslydescribed embodiments can be combined in any manner with one or morefeatures of any other embodiments in the present invention. Furthermore,many variations of the invention will become apparent to those skilledin the art upon review of the specification. The scope of the inventionshould, therefore, be determined not with reference to the abovedescription, but instead should be determined with reference to theappended claims along with their full scope of equivalents.

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By their citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

1. A method of diagnosing minimal change nephrotic syndrome in a patienthaving kidney disease, the method comprising: (a) obtaining a samplefrom an idiopathic nephrotic syndrome patient; (b) detecting alpha-2-HSglycoprotein beta chain in the sample from the patient; and (c)correlating elevation of the alpha-2-HS glycoprotein in the sample witha control amount to diagnose minimal change nephrotic syndrome.
 2. Themethod of claim 1, wherein detection is performed by immunoassay.
 3. Themethod of claim 1, wherein detection is performed by SELDI MS massspectrometry.
 4. The method of claim 1 where the sample is blood serum.